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<p><b>BACKGROUND</b>Injectable three-dimensional (3D) scaffolds have the advantages of fluidity and moldability to fill irregular-shaped defects, simple incorporation of bioactive factors, and limited surgical invasiveness. Adipose-derived stem cells (ADSCs) are multipotent and can be differentiated toward nucleus pulposus (NP)-like cells. A hypoxic environment may be important for differentiation to NP-like cells because the intervertebral disc is an avascular tissue. Hence, we investigated the induction effects of hypoxia and an injectable 3D chitosan-alginate (C/A) gel scaffold on ADSCs.</p><p><b>METHODS</b>The C/A gel scaffold consisted of medical-grade chitosan and alginate. Gel porosity was calculated by liquid displacement method. Pore microstructure was analyzed by light and scanning electron microscopy. ADSCs were isolated and cultured by conventional methods. Passage 2 BrdU-labeled ADSCs were co-cultured with the C/A gel. ADSCs were divided into three groups (control, normoxia-induced, and hypoxia-induced groups). In the control group, cells were cultured in 10% FBS/DMEM. Hypoxia-induced and normoxia-induced groups were induced by adding transforming growth factor-β1, dexamethasone, vitamin C, sodium pyruvate, proline, bone morphogenetic protein-7, and 1% ITS-plus to the culture medium and maintaining in 2% and 20% O2, respectively. Histological and morphological changes were observed by light and electron microscopy. ADSCs were characterized by flow cytometry. Cell viability was investigated by BrdU incorporation. Proteoglycan and type II collagen were measured by safranin O staining and the Sircol method, respectively. mRNA expression of hypoxia-inducing factor-1α (HIF-1α), aggrecan, and Type II collagen was determined by reverse transcription-polymerase chain reaction.</p><p><b>RESULTS</b>C/A gels had porous exterior surfaces with 80.57% porosity and 50-200 üm pore size. Flow cytometric analysis of passage 2 rabbit ADSCs showed high CD90 expression, while CD45 expression was very low. The morphology of induced ADSCs resembled that of NP cells. BrdU immunofluorescence showed that most ADSCs survived and proliferated in the C/A gel scaffold. Scanning electron microscopy showed that ADSCs grew well in the C/A gel scaffold. ADSCs in the C/A gel scaffold were positive for safranin O staining. Hypoxia-induced and normoxia-induced groups produced more proteoglycan and Type II collagen than the control group (P < 0.05). Proteoglycan and Type II collagen levels in the hypoxia-induced group were higher than those in the normoxia-induced group (P < 0.05). Compared with the control group, higher mRNA expression of HIF-1α, aggrecan, and Type II collagen was detected in hypoxia-induced and normoxiainduced groups (P < 0.05). Expression of these genes in the hypoxia-induced group was significantly higher than that in the normoxia-induced group (P < 0.05).</p><p><b>CONCLUSION</b>ADSCs grow well in C/A gel scaffolds and differentiate toward NP-like cells that produce the same extracellular matrix as that of NP cells under certain induction conditions, which is promoted in a hypoxic state.</p>
Subject(s)
Animals , Rabbits , Adipose Tissue , Cell Biology , Alginates , Chemistry , Cell Differentiation , Physiology , Cells, Cultured , Chitosan , Chemistry , Glucuronic Acid , Chemistry , Hexuronic Acids , Chemistry , Stem Cells , Cell Biology , Physiology , Tissue Engineering , Methods , Tissue Scaffolds , ChemistryABSTRACT
Objective To explore the strategy and outcomes of surgical treatment of thoracic ossification of ligamentum flavum(OLF),especially combined with ossification of posterior longitudinal ligament,thoracic kyphosis and epidural adhesion.Methods Fifty-three cases of thoracic OLF from January 2003 to December 2009 were reviewed retrospectively.All patients were treated by the methods of en bloc resection of semi-facet and lamina.All patients were followed up for more than half an year,including 32 males and 21 females,aged from 43 to 73 years(average 54.7 years).The lesions located in upper thoracic for 18 patients,and in thoracolumbar for 35 patients.For multi-level or jumping OLF patients,the responsible levels were determined by combination of images and clinical symptoms.For multi-level OLF with ossification of posterior longitudinal ligament(OPLL)or thoracic kyphosis(＞50°),multi-level pedicle screw fixation and correction of kyphosis were performed.For dural adhesion patients,part of cerebrospinal fluid was released with a caudal incision of dural sac resulting in collapse and epidural arachnoid separation.Ossific and adhesion dura mater were removed with integrity of arachnoid.The surgical outcomes were evaluated with preoperative and postoperative thoracic Japanese Orthopaedic Association(JOA)score,Nurick grade and neurologic functional recovery ratio.Results Fifty-three cases were followed up for 6 months to 6 years,with an average of 18 months.The average preoperative JOA score was 4.3±2.3,which significantly increased to 8.3±1.8 after operation.Postoperative neurologic functional recovery rates were 11% to 80%(average 65.8%),including excellent in 18 cases,good in 20,fair in 10,and poor in 5.The excellent or good rate was 71.7%.The mean preoperative Nurick grade was 3.7(2-5 grade)and decreased to 2.3 grade after operation.Conclusion En bloc resection of semi-facet and lamina is a safe and effective method for treatment of thoracic OLF.For the patients with OPLL or kyphosis,pedicle screws fixation and kyphosis correction was beneficial for recovery of neurologic function of thoracic OLF patients.
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@#Objective To investigate the osteogenic potential for size-critical bone defect of fibrin sealant combined with recombined human bone morphogenetic protein-2 (rhBMP-2) grafting and varied autologous bone marrow mesenchymal stem cells (BMSCs) implanting in vivo. Methods BMSCs were cultured and induced with osteogenic supplement (OS) medium. BMSCs with and without OS induction were collected and percutaneously autologous injected respectively into the 15 mm bone defect of experimental rabbit model. The grafts were BMSCs, osteo-induced BMSCs, BMSCs and osteo-induced BMSCs, BMP combined with fibrin sealant, 0.9% NaCl solution. Osteogenesis at the defect area was assessed with regular radiography, histology and biomechanics. Results The FS/BMP group and the BMSCs+osteo-induced BMSCs group achieved complete bone healing with medullary cavity united, with the most new bone formation and the maximal load among those groups. Conclusion The osteogenic potential of both osteo-induced BMSCs combined with BMSCs and FS/BMP are similar, which are superior to that of BMSCs or osteo-induced BMSCs along.
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@#Objective To investigate the clinical characteristics of postoperative spondylodiscitis after lumbar intervertebral disc herniation.Methods 9 cases with spondylodiscitis were reviewed,about their clinical symptoms,laboratory tests,bacteria culture,CT and MRI finging,etc.Results and Conclusion The clinical symptoms occurred 8~24 d after operation.Laboratory test and MRI were helpful for the early diagnosis.The outcome of both surgical and non-surgical treatment was satisfactory.
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@#Objective To explore the changes of rabbit adipose stem cells(ASCs)and bone mesenchymal stem cells(BMSCs)cultured in vitro and the anabolism under basic fibroblast growth factor(bFGF).Methods BMSCs and ASCs were cultured with DMEM,DMEM/F12(2∶1)or α-MEM respectively.The 3rd generation ASCs and BMSCs were divided into 2 groups respectively:group A:ASCs cultured in chondrogenic medium(CM),group B:ASCs cultured in CM supplemented with bFGF 5 ng/ml,group C:BMSCs cultured in CM,group D:BMSCs cultured in CM supplemented with bFGF 5 ng/ml.Morphological changes were observed under inverted microscope.The 35SO42-incorporation and total hydroxyproline were measured.Results BMSCs and ASCs showed much higher growth rate when cultured in α-MEM medium comparison with that in DMEM or in DMEM/F12(2:1).Both stem cells attachment cultured in monolayer greatly increased and cell clones were abundant,while the cells attachment became rather difficult and cell clones were less after cutured in CM.All stem cells possessed a round-like morphology,and the cells in group B and D were more than that in the other 2 groups.The 35SO42-incorporation and total hydroxyproline synthesis of group B or D increased compared with that of group A or C,but there was no diference between group D and B.Conclusion The rabbit ASCs and BMSCs cultured in CM suppling with bFGF grow well and their metabolism increased.
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@#Objective To explore the mechanism of percutaneous laser disc decompression (PLDD) for treating prolapse of intervertebral disc. Methods 15 rabbits were divided into 3 groups randomly: sham group, model group with PLDD treatment, model group without PLDD treatment. The nerve conduction velocity (NCV) of L6 nerve root and the activity of phospholipase A2 (PLA2) in the intervertebral disc of L5-6 were determined 2 weeks after the initial surgery. Results NCV in the group with PLDD was significantly faster than that in the group without PLDD (P<0.001); NCV in the group without PLDD was significantly lower than that in the sham group (P<0.001). The activity of PLA2 in the group with PLDD was significantly lower than that in the group without PLDD (P<0.001); The activity of PLA2 in the group without PLDD was significantly higher than that in the sham group (P<0.001). Conclusion The activity of PLA2 in the herniated discs is higher than that in normal discs, which result in NCV falls remarkably. The PLDD can reduces chemical factors such as PLA2.
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@#ObjectiveTo analysis the clinical features of thoracic spine and spinal cord injury (SCI) and summarize the inclusive standard of cellular transplant clinical trial for SCI.MethodsThe data of 72 cases with thoracic spine and spinal cord injury from 1990 to 2005 were analyzed retrospectively.ResultsMean follow-up period was 20 months (6～48 months). There was no recovery in 12 spinal cord injury without radiographic abnormality (SCIWORA) patients, but improvement of urine function in 4 cases. 5 cases of 52 fracture-dislocation complete injury were improved to grade B (sense recovery), rate of recovery was 9.6%; recovery rate was 62.5% in incomplete injury. Sense recovery of all cases was better than motor recovery. Partial cases appeared spasm paralysis relief.ConclusionIncidence rate of complete injury is high and recovery is bad in thoracic spine and spinal cord injury. The inclusive standard of cellular transplant clinical trial for SCI is old complete thoracic spinal cord injury without residual compression.